Theory and simulation of DNA repair enzymes; mechanism, structure and function Project Summary Human genome integrity depends on many processes to ensure the fidelity of the duplication of DNA.The efficiency of these processes is crucial since errors in DNA can often be key to disease onset. An important process to insure genome integrity is the repair of damaged DNA.[1] There are several types of DNA damage including (but not limited to): alkylation, oxidation, hydrolysis, adduct formation, base mismatch, among others. Alkylated DNA bases may be removed by two main routes: excision of the damaged base and activation of the base excision repair (BER) process, or direct dealkylation. The former route involves several enzymes involved in the BER cascade. The latter route may be performed by the AlkB family of enzymes. AlkB family enzymes are non-heme iron and ?-ketoglutarate dependent enzymes that perform an oxidative dealkylation of DNA. Some cancer treatments involve alkylating agents, and attempts have been made to enhance these therapies by inhibiting alkylating damage repair. Information gained from a detailed understanding of the structure and reaction mechanism of AlkB family proteins can aid in the development of inhibitors for these enzymes by providing useful information to develop transition state analogue inhibitors. One approach for this is via computational methods, including quantum mechanical/molecular mechanical (QM/MM) methods. Currently, most QM/MM implementations employ force fields that may not accurately describe the MM environment at close range, are not polarizable and lack methods to include long-range electrostatic effects. Our long-term goal is to understand the mechanism, structure and function of enzymes involved in DNA repair by means of computational simulations. To this end, the goals of GM108583 are: i) To study the structure/function/reactivity of AlkB family of enzymes by quantum mechanical/molecular mechanical (QM/MM), molecular dynamics (MD) and homology modeling. ii) To develop the first QM/MM program that interfaces a QM program with a two advanced force fields (GEM and AMOEBA) to accurately describe the MM environment; and to develop a novel method to introduce long-range electrostatic effects in QM/MM simulations. The detailed understanding of the structure, function and reaction mechanism of AlkB and its human homologues will provide insights into possible methods to inhibit these enzymes. In the current funding cycle we have already developed LICHEM [2] and pmemd.gem [3], collaborated in the development of TINKER?HP [4], and created QM/MM?LREC for long?range electrostatics in QM/MM [5, 6]. We reported new insights on ALKB enzymes including detailed understanding of the reaction mechanism of AlkB [7, 8]; confirmed an intra?molecular O2 tunnel in AlkB [9], and a homology model for ALKBH1 [10]. We used our HyDn?SNP?S method to uncover the first ever biomarker for prostate cancer on ALKBH7, and computationally predicted it?s effect on substrate binding, which was confirmed experimentally by our collaborators [11]. In addition we established collaborations with other experimental groups to investigate different DNA modification enzymes [12?15] and to improve force fields for computational simulations [16?20]. We are continuing our investigation of the reaction mechanism of ALKBH2 and ALKBH3 as well as expanding our investigation of enzymes of this family as well as other related enzymes based on our newly established collaborations.